1. Area of the Art
The present invention concerns the art of microfluidics—namely the transporting and processing of microliter—volume fluid samples. More specifically, this invention addresses the problem of improving biosensor detection by processing the sample volume in the form of discrete mobile droplets which can then be agitated, evaporated or driven over one sensor or a plurality of sensors.
2. Description of the Prior Art
A key performance criterion of many biosensors is their ability to detect low concentrations of target biomolecules (by biomolecules we mean proteins, nucleic acids, polysaccharides, lipids and other organic molecules typical of living organisms; in some cases the target consists of small particles—e.g. cellular organelles or fractions—instead of individual biomolecules). This task is made more difficult because 1) the specific target biomolecules are almost always randomly distributed throughout the sample volume and not concentrated at the sensing site; 2) the presence of other non-target or non-specific molecules within the sample can degrade the sensor response, leading to either false positives or to masking target biomolecule detection in by background noise; and 3) the actual detection of captured target molecules may require application of secondary markers and/or reaction substrates to the sensor.
Rather than relying solely on random diffusion effects to bring the target molecules into proximity to the sensor, a variety of sample processing tactics have been employed to improve biosensor detection. These techniques include: 1) concentrating or focusing target molecules at the sensing site(s); 2) circulating, or agitating the sample volume at the sensing site(s); and 3) providing reaction substrates or other materials which enable or amplify detection. These methods are currently used in biosensor, lab-on-chip, micro-total-analysis, microarray and other microfluidic applications; however each of these techniques has its own drawbacks.
Focusing or concentrating target molecules within the sample volume via electrophoretic or other affinity means can also concentrate interfering non-target molecules which are attracted by the same effects intended to concentrate the target molecules. Therefore, a way to wash the sensor site, typically achieved by valving and pumping, must also be included to remove such non-target molecules. Generally, the target molecules will interact with the sensing site and resist removal by washing. Concentration can also fail when some or all of the target molecules are not sufficiently charged to be concentrated by electrophoresis and/or lack characteristics to be concentrated by the affinity method employed.
Rather than focusing by means of electrophoresis or otherwise concentrating target molecules from within the sample volume, some techniques seek to improve the probability of detection by circulating or agitating the entire sample volume over the sensing site. Circulation and agitation effects not only bring new target molecules into contact with the sensing site, but also serve to wash or remove non-target molecules from the site. However, to circulate or agitate the entire sample volume, cumbersome seals, valving and costly mechanical, pneumatic, or surface acoustic wave methods are typically required. Such complexity would likely impede application of such techniques to systems including numerous and/or highly distributed biosensors.
Once the target molecules have been captured by the sensing site, it is often necessary to treat the captured target molecules at the sensing site with a labeling or reaction solution to enable detection, amplify detection, and/or quantify the captured target molecules. For example, an antibody that has been fluorescently labeled and which is specific to the target molecule can be introduced to the sensing site to create an optical signature proportional to the number of captured target molecules. For some electronic sensors, a substrate solution that specifically reacts with the captured target molecule (or enzymes linked to the target molecule) thereby releasing molecules directly detectable by the sensor can be introduced to create an electronic signal that is proportional to the number of captured target molecules. However in either case, when the indicator or marker signal changes with time, precise timing is required in the delivery of the reaction solution(s) to achieve accurate comparisons. Unfortunately, leaky valving, air bubbles in channels, variations in capillary wetting characteristics and even differences in manual loading of samples and reagents often compromise precision timing of microfluidic delivery of such reaction solution(s).